U.S. patent number 5,122,594 [Application Number 07/379,002] was granted by the patent office on 1992-06-16 for modified human pancreatic secretory trypsin inhibitor.
This patent grant is currently assigned to Shionogi & Co., Ltd.. Invention is credited to Norihisa Kikuchi, Masaru Shin, Hiroshi Teraoka, Nobuo Yoshida.
United States Patent |
5,122,594 |
Yoshida , et al. |
June 16, 1992 |
Modified human pancreatic secretory trypsin inhibitor
Abstract
DNA sequences encoding modified varieties of human PSTI
possessing excellent stability in terms of decreased susceptibility
to decomposition by proteolytic enzymes such as trypsin, as
compared with natural human PSTI, as well as the modified varieties
of human PSTI obtained by the expression of the DNA sequences.
Inventors: |
Yoshida; Nobuo (Nishinomiya,
JP), Kikuchi; Norihisa (Takatsuki, JP),
Shin; Masaru (Kobe, JP), Teraoka; Hiroshi (Sakai,
JP) |
Assignee: |
Shionogi & Co., Ltd.
(Osaka, JP)
|
Family
ID: |
26500552 |
Appl.
No.: |
07/379,002 |
Filed: |
July 12, 1989 |
Foreign Application Priority Data
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Jul 19, 1988 [JP] |
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63-181316 |
Oct 11, 1988 [JP] |
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63-255580 |
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Current U.S.
Class: |
530/324;
435/69.2; 530/845; 536/23.5; 435/320.1 |
Current CPC
Class: |
C07K
14/8135 (20130101); C12N 9/12 (20130101); Y10S
530/845 (20130101); A61K 38/00 (20130101); C07K
2319/00 (20130101) |
Current International
Class: |
C07K
14/81 (20060101); C12N 9/12 (20060101); A61K
38/00 (20060101); C07K 007/10 (); C12N
015/15 () |
Field of
Search: |
;530/324,845
;435/69.2,320.1 ;536/27 |
Foreign Patent Documents
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0264118 |
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Apr 1988 |
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EP |
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63-267289 |
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Nov 1988 |
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JP |
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2199582A |
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Jul 1988 |
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GB |
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Other References
L J. Greene et al. Methods Enzymol. vol. 45, 1976, pp. 813-825.
.
T. Yamamoto et al. Biochemical and Biophysical Research
Communications, vol. 132, No. 2, Oct. 30, 1985, pp. 605-612. .
M. H. Pubols et al. The Journal of Biological Chemistry, vol. 249,
No. 7, Apr. 10, 1974, pp. 2235-2242. .
G. Feinstein et al. Eur. J. Biochem. 43, 1974 pp. 569-581..
|
Primary Examiner: Lacey; David L.
Assistant Examiner: Ossanna; Nina
Attorney, Agent or Firm: Morrison & Foerster
Claims
What is claimed is:
1. A modified human pancreatic secretory trypsin inhibitor in which
the arginines at the 42nd and/or 44th positions from the N-terminus
of the amino acid sequence of natural human pancreatic secretory
trypsin inhibitor are replaced by glutamine and/or serine.
2. A DNA sequence encoding the modified human pancreatic secretory
trypsin inhibitor set forth in claim 1.
3. A modified human pancreatic secretory trypsin inhibitor in which
the arginine at the 42nd or 44th position from the N-terminus of
the amino acid sequence of natural human pancreatic secretory
trypsin inhibitor is replaced by glutamine or serine,
respectively.
4. A DNA sequence encoding the modified human pancreatic secretory
trypsin inhibitor set forth in claim 3.
Description
BACKGROUND OF THE INVENTION
1. Field of the invention:
This invention relates to varieties of modified human PSTI and to
DNA sequences encoding the same.
2. Description of the prior art:.
Two types of trypsin inhibitor are known which are derived from the
pancreas, i.e., pancreatic secretory trypsin inhibitor (PSTI) and
basic pancreatic trypsin inhibitor (BPTI). PSTI is present in all
mammals, and is distributed not only in the pancreas but also in
the kidney, lung, spleen, liver, brain and other organs. BPTI is
distributed in various viscera of cows and other ruminants, but is
not present in man or other mammals. Pubols et al. (J. Biol. Chem.
249, 2235, 1974) and Feinstein et al. (Eur. J. Biochem. 43, 569,
1974) have isolated and purified PSTI from human pancreatic juice,
and Greene et al. (Methods Enzymol. 45. 813, 1976) determined the
structure of this substance. Furthermore, Yamamoto et al. (Biochem.
Biophys. Res. Commun. 132, 605, 1985) determined the DNA sequence
corresponding to PSTI (FIG. 5). As shown by FIG. 5, human PSTI is a
peptide composed of 56 amino acid residues, with a molecular weight
of 6,242 daltons. It is known that sulfhydryl groups do not exist
in PSTI, since the cysteine residues at positions 9 and 38, as well
as 16 and 35, and also 24 and 56 are linked by disulfide bonds.
The trypsin inhibitor described above is present in the acinic
cells of the pancreas, and in normal humans is secreted in the
pancreatic juice together with various pancreatic enzymes so that
it inhibits trypsin in the ductus pancreaticus. However, in acute
pancreatitis, for some reason trypsin is activated and then
trypsinogen and other enzyme precursors are activated in a chain
reaction, and this presumably results in autodigestion of the
pancreas. The administration of trypsin inhibitor is effective in
the therapeutic treatment of this type of acute pancreatitis. The
trypsin inhibitors currently used for this purpose include the
above-mentioned bovine pancreatic BPTI as well as synthetic
inhibitory agents, etc. In view of its source, human PSTI would
appear to be the most appropriate trypsin inhibitor for use in this
sort of therapy. However, since this form of PSTI has heretofore
been prepared by isolation and purification from human pancreatic
juice, sufficiently large quantities for therapeutic use could not
be obtained, and therefore up to the present time human PSTI has
not been employed in clinical practice. In order to solve this
problem of quantitative production, the present inventors have
developed a method of obtaining large quantities of human PSTI by
applying recombinant DNA techniques (Japanese Laid-Open Patent
Publication No. 62-253437). According to this method, human PSTI is
expressed as a fusion protein with APH (aminoglycoside
3'-phosphotransferase II). This human PSTI fusion protein can be
produced in large quantities in a microbial host, and after
cleavage of this fusion protein with cyanogen bromide, human PSTI
alone can be isolated and purified. The human PSTI obtained by this
method possesses the same amino acid sequence as natural human
PSTI, and therefore one may expect the same degree of therapeutic
efficacy as that obtainable with natural PSTI in clinical
applications. However, PSTI is also a peptide, and therefore with
passage of time PSTI is gradually decomposed by proteolytic enzymes
such as trypsin. Owing to this shortcoming, in order to achieve an
adequately sustained trypsininhibiting effect, the quantity of PSTI
which decomposes with passage of time must be monitored and
replaced by an equal amount of the fresh substance, which has
necessitated troublesome laboratory testing and other additional
procedures.
SUMMARY OF THE INVENTION
The inventors have discovered that, by introducing site-specific
mutations into the gene which encodes human PSTI, varieties of
human PSTI (modified PSTI) with characteristics different from
those of the naturally occurring form of PSTI (natural PSTI) can be
obtained, and thereby succeeded in completing the present
invention.
A modified human PSTI of the present invention, which overcomes the
above-discussed and numerous other disadvantages and deficiencies
of the prior art, is identical with natural human PSTI except that
the arginines at the 42nd and/or 44th positions from the N-terminus
of the amino acid sequence of the natural human PSTI are replaced
by glutamine and/or serine.
A DNA sequence of the present invention encodes the above-mentioned
modified human PSTI derived by replacing the arginines in the
number 42 and/or 44 position from the N-terminus of the amino acid
sequence of natural human PSTI by glutamine and/or serine.
Another modified human PSTI of the present invention is identical
with natural human PSTI except that the arginine at the 42nd or
44th position from the N-terminus of the amino acid sequence of the
natural human PSTI is replaced by glutamine or serine,
respectively.
Another DNA sequence of the present invention encodes the
above-mentioned modified human PSTI derived by replacing the
arginine in the number 42 or 44 position from the N-terminus of the
amino acid sequence of natural PSTI by glutamine or serine,
respectively.
Thus, the invention described herein makes possible the objectives
of (1) providing modified varieties of human PSTI; which are more
resistant to decomposition by trypsin and other proteolytic enzymes
than natural human PSTI and (2) providing DNA sequences which
encode modified human PSTI with the advantageous properties stated
above by the application of recombinant DNA techniques.
DETAILED DESCRIPTION OF THE INVENTION
The DNA sequences encoding the modified human PSTI of the present
invention can be obtained, for example, by using recombinant DNA
techniques, specifically, by preparing an expression vector which
has the gene encoding natural human PSTI (obtainable by the method
described by the inventors in Japanese Laid-Open Patent Publication
No. 62-253437) downstream from a suitable promoter, and then
introducing a site-specific mutation into the human PSTI gene in
this vector. Since the amino acid sequence of human PSTI is
comparatively short, the desired variety of modified PSTI can also
be obtained by direct chemical synthesis. However, once a
recombinant possessing the gene which encodes human PSTI has been
prepared, the introduction of a site-specific mutation into this
vector to obtain the gene encoding the desired modified PSTI is
easily effected, and therefore this method is highly appropriate
for the purpose in view. The gene encoding human PSTI has already
been cloned from human pancreatic cells by Yamamoto et al. (v.s.),
and the DNA sequence of this gene has also been determined. This
DNA can also be prepared from human pancreatic cells in accordance
with the procedure of Yamamoto et al., but since this sequence is
comparatively short, the use of synthetic human PSTI genes is
advantageous. The DNA sequence of natural human PSTI is shown in
FIG. 5. In the present invention, any DNA sequence encoding the
amino acid sequence of human PSTI shown in FIG. 5 can be used. This
human PSTI gene is converted into a fusion gene with another gene
which possesses highlevel expressive capability under control of a
suitable promoter. For example, this can be appropriately
accomplished by the formation of a fusion gene with an APH gene in
accordance with the method of the above-cited Japanese Laid-Open
Patent Publication No. 62-253427. Here, the term APH gene refers to
one which contains the structural gene encoding APH
(amino-glycoside 3'-phosphotransferase II), and may also contain a
promoter, etc. APH genes confer drug resistance against neomycin
and kanamycin upon microorganisms.
The base sequence of this APH gene has already been known (Gene,
19, 327, 1982). This base sequence and the amino acid sequence
deduced from this base sequence are shown in FIG. 6. A transposon
Tn5 and plasmids (e.g., pNEO (Pharmacia)) containing this base
sequence are commercially available, and APH genes can be obtained
by excision from these element. These APH genes need not contain
the complete structural gene for natural APH, and need only encode
several amino acids at the N-terminus. For example, one may use the
restriction fragment of pNEO (Pharmacia) digested by HindIII and
TaqI (containing the APH promoter and the gene encoding the amino
acid sequence from the N-terminus to the 82nd amino acid of APH,
corresponding to the DNA sequence from the -350 to the 246 position
in FIG. 6). Moreover, not only the sequence shown in FIG. 6, but
also any modified APH sequence derived from this by substitution,
deletion or insertion of some nucleotides can be used.
In order to obtain a gene encoding the modified human PSTI of the
present invention, one can, for example, first synthesize the DNA
sequence encoding human PSTI. Such a DNA sequence can be
synthesized, for example, by synthesizing the 20 types of fragments
(U-1 to U-10 and L-1 to L-10) shown in FIG. 7 with the use of an
automatic nucleic acid synthesizer, then purifying these products
by a chromatographic technique such as high performance liquid
chromatography, and after attaching phosphate residues to all these
fragments except U-1 and L-10, appropriately joining the fragments
with DNA ligases. This type of method is described in Nucleic Acids
Res. 13, 2959 (1985). After ligation, the DNA is recovered as usual
by phenol extraction and ethanol precipitation, and then
fractionated by a conventional method such as polyacrylamide gel
electrophoresis. The recovery of the desired DNA fraction from the
polyacrylamide gel can be accomplished, for example, by adsorption
and elution using a DEAE-C membrane, as described in "Molecular
Cloning" (Cold Spring Harbor Laboratory, New York, 1982).
In order to determine the base sequences of the recovered DNA
fragments, one may, for example, insert these DNA fragments into an
M13 phage vector, use this to transform a suitable host, and then
apply screening procedures. M13 phage vectors suitable for this
purpose include M13mplO (manufactured by Takara Shuzo Co.). By
cleaving this phage vector with appropriate restriction
endonucleases and joining the cleaved vector to the above-mentioned
DNA fragments with T4 DNA ligase, one constructs a recombinant
phage M13-PSTI, which incorporates DNA encoding human PSTI. This
M13-PSTI phage is then introduced into an appropriate host cell.
This can be accomplished by, for example, the method described in
"Molecular Cloning" (v.s., pp. 250-251). One host cell appropriate
for this purpose is Escherichia coli K-12 strain JM103. The
bacteria into which M13mp10 have been introduced from blue plaques,
whereas bacteria transformed by the introduction of M13-PSTI form
colorless plaques. If E. coli is transfected by the phage DNA
obtained from the colorless plaques, this phage DNA proliferates in
the bacterial culture, and single-stranded phage DNA is obtained
from the supernatant of the culture medium while double-stranded
phage DNA can be obtained from the bacterial cell bodies. The
single-stranded DNA can be prepared by the method of Messing
(Methods Enzymol. 101, 20-28 (1983)). By applying the dideoxy
method of base sequencing (Science 214, 1205.congruent.1210 (1981))
to the single-stranded DNA, one can determine whether or not the
desired complete structural gene for human PSTI has been inserted.
This is a general method, specifically, for example, the
commercially marketed M13 Sequencing Kit (manufactured by Takara
Shuzo Co.) can be utilized. The preparation of double-stranded DNA
from the bacterial cell bodies can be accomplished by using the
conventional sodium hydroxide-sodium dodeoylsulfate (SDS) method
(Nucleic Acids Res. 7, 1513-1523 (1979)). The double-stranded DNA
obtained by this method is used in the construction of expression
plasmids.
The PSTI gene is excised from the M13 phage recombinant obtained in
this manner, and this gene together with an APH gene excised from
the aforementioned pNEO or other vector is inserted into an
appropriate plasmid vector, resulting in the desired PSTI
expression plasmid. In doing this, the presence of the codon for
methionine (i.e., ATG) at the 5' end of the above-mentioned human
PSTI gene sequence is desirable. If a gene which encodes a fusion
protein with methionine located between the APH and human PSTI
moieties is constructed in this manner, then the linkage between
the APH and the PSTI can be cleaved by treating the expressed
fusion protein with cyanogen bromide, thus facilitating the
isolation of human PSTI. The expression plasmid (pUC13-PSTI) can be
constructed, for example, by ligating 1) a 180 bp DNA fragment
obtained by cleaving the above-mentioned doublestranded DNA with
AccI and BamHI, 2) the approximately 2.8 kbp DNA fragment obtained
by HindIII-BamHI cleavage of pUC13 and 3) the approximately 600 bp
DNA fragment obtained by digestion of pNEO (containing the APH gene
of Tn5) with HindIII and TaqI (pUC13-PCTI). In addition to the
pUC-13 mentioned in 2) above, other plasmid vectors which can be
employed for this construction include p.beta.-ga113C, pOP203-13,
pUC9, pUC8, pEA300, ptrpLI, pBN70, pWTIII, pWT121, pWT131,
pKK223-3, pDR540, pDR720, pYEJOOI, pPL-lambda, pKC30, pKC31 , pASl,
pLC24, pHUB4, pIN-I, pIN-II, pIN-III, pC194, pC221, pUB112, pT127,
pSA0503, pE194, etc.; however, the possibilities are not confined
to this list; in fact, provided only that the above-described human
PSTI and APH fusion gene can be transferred by the vector and
expressed in some microorganism, any of the vectors generally
employed for transformation by those skilled in genetic engineering
can be used for the present purpose. By selecting a vector
appropriate for the host, and situating the above-described fusion
gene under the control of a suitable promoter, one can construct a
recombinant capable of expressing the required APH-human PSTI
fusion protein. A promoter for the APH gene is contained in the DNA
fragment mentioned in 3) above, obtained by digestion of pNEO;
however, this promoter may be changed into another promoter, or the
APH gene may placed downstream from an even stronger promoter. The
promoters which can be used for the present purpose are the lac,
Trp, Tac promoter systems, etc.
The expression plasmid obtained from the above-mentioned DNA
fragments 1), 2) and 3) can be introduced into a suitable host and
checked for production of PSTI. For example, expression of PSTI as
a fusion protein with APH can be verified by transforming suitable
host cells through the introduction of the above-mentioned
expression plasmid pUC13-PSTI in accordance with the method
described in "Molecular Cloning" (v.s.). If host cells such as E.
coli (i.e., K-12 strain JM103, C600, AG-1, etc.) or B. subtilis are
employed, then PSTI can be produced with high efficiency. Since
natural human PSTI has no sugar chains, human PSTI of the same type
as the natural form can be produced in prokaryotic cells. The
transformed cells are selected for ampicillin resistance. Then, the
plasmids contained in these cells are cleaved with HindIII, BamHI
and PstI, then analyzed by the sodium hydroxide-SDS method, and the
plasmids which are obtained as approximately 3.6 kbp DNA bands are
selected. Host cells containing the plasmids selected in this
manner are cultured in the presence of ampicillin, the bacterial
cell bodies are collected, solubilized and analyzed by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the
detection of a band corresponding to the PSTI-APH fusion protein,
with a molecular weight of 15,000 daltons, verifies that the gene
for this fusion protein is indeed being expressed in the host
cells.
In order to obtain human PSTI from the fusion protein, since
methionine has been inserted between the two proteins as indicated
above, human PSTI is easily separated by treatment with cyanogen
bromide. Other methods which can ordinarily be employed for this
separation include insertion of cysteine between the two proteins
and subsequent cleaving with 2-nitro-5-thiocyanobenzoic acid,
insertion of asparagine-glycine therebetween and subsequent
cleaving with hydroxylamine, insertion of tryptophan therebetween
and subsequent cleaving with
2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindole, insertion of
lysine or arginine therebetween and subsequent cleaving with
trypsin, insertion of the sequence isoleucine-glutamic
acid-glycine-arginine therebetween and subsequent cleaving with
blood coagulation factor Xa, etc.; taking the amino acid sequence
of human PSTI into consideration, these various methods can be used
under appropriate circumstances.
The human PSTI which has been cleaved from the fusion protein can
be purified in the usual manner by an appropriate combination of a
chromatographic process such as gel filtration chromatography or
affinity chromatography, centrifugal separation, etc. Amino acid
analysis of the purified PSTI has verified that the amino acid
composition of the product is indeed completely identical with that
of natural human PSTI (Example 1, Table 1).
Next, in order to obtain the modified human PSTI of the present
invention, the expression plasmid pUC13-PSTI described above is
used to introduce a site-specific mutation into the PSTI gene. In
this manner, one obtains a recombinant possessing DNA which encodes
the desired modified human PSTI. This site-specific mutagenesis is
effected by an ingenious combination of chemical techniques for DNA
synthesis and the enzymatic reactions of DNA replication. To
perform this processing, first, one employs chemical methods to
synthesize oligonucleotides (short DNA fragments) such that only
the base at the target position in the DNA sequence has been
altered and the remaining bases are complementary to those of the
desired DNA sequence. These DNA fragments are then paired with the
DNA which is to undergo mutation (prepared beforehand in
single-stranded form). Then, by subjecting these fragments to the
action of DNA polymerase, one can synthesize DNA which contains the
chemically synthesized oligonucleotides with the altered base
sequence and is complementary to the original DNA at all other
positions. That is, any DNA molecule with mutations introduced at
desired locations can be synthesized in this manner. Specifically,
in order to prepare a recombinant possessing DNA which encodes
modified human PSTI by the above-described method of site-specific
mutagenesis, for example, one first cleaves pUC13-PSTI with
restriction endonucleases such as HindIII and BamHI, thus obtaining
a fusion gene for human PSTI and APH. This is ligated to the M13
phage vector M13mp10, thereby preparing the recombinant phage
M13-APH/PSTI. On the other hand, one also chemically synthesizes
the oligonucleotides indicated by the formulae (1) to (3) below,
using an automatic nucleic acid synthesizer. ##STR1##
Each of the synthesized oligonucleotides (1) to (3) is then
purified by an appropriate combination of chromatographic methods
such as gel filtration, high performance liquid chromatography,
etc. These purified synthetic oligonucleotides are then
phosphorylated and annealed to the above-mentioned recombinant
M13-APH/PSTI (which has previously been prepared in single-stranded
form). From this annealed hybrid DNA, double-stranded DNA is
prepared by using Klenow fragment (Klenow enzyme) and DNA ligase,
and the unreacted single-stranded DNA is removed with a
nitrocellulose filter, etc. From the double stranded DNA obtained
in this manner, one can prepare a recombinant possessing DNA which
encodes the modified human PSTI (Ser(44)-PSTI, Gln(42)-PSTI or
Thr(43)-PSTI).
By using the dideoxy method to determine the DNA sequence of the
modified PSTI gene contained in these recombinants, one may verify
that entire sequence of the structural gene for the desired
modified human PSTI is included. Employing this method, the
inventors have successfully obtained the following DNA sequences
(a)-(c).
(a) A DNA sequence identical with that encoding human PSTI except
that the guanine residue at the number 125 position from the 5' end
has been replaced by adenine (corresponding to a peptide
Gln(42)-PSTI derived from PSTI by replacing the arginine at the
42nd position from the N-terminus by glutamine).
(b) A DNA sequence identical with that encoding human PSTI except
that the cytosine residue at the number 130 position from the 5'
end has been replaced by adenine (corresponding to a peptide
Ser(44)-PSTI derived from PSTI by replacing the arginine at the
44th position from the N-terminus by serine).
(c) A DNA sequence identical with that encoding human PSTI except
that the adenine residue at the number 128 position from the 5' end
has been replaced by cytosine (corresponding to a peptide
Thr(43)-PSTI derived from PSTI by replacing the lysine at 43rd
position from the N-terminus by threonine).
The base sequences and corresponding amino acid sequences of two of
these products, i.e., Gln(42)-PSTI and Ser(44)-PSTI, are shown in
FIGS. 1 and 2, respectively.
Next, an expression plasmid is constructed in order to express the
modified human PSTI obtained by the above method. To accomplish
this, first, the above-described recombinant containing the fusion
gene encoding APH and modified human PSTI is treated with the
restriction enzymes EcoRI and HindIII, thereby excising the said
fusion gene. This DNA fragment is isolated by a method such as
polyacrylamide gel electrophoresis and inserted into a suitable
plasmid vector. Any of the previously mentioned plasmids used as
expression vectors for human PSTI can also be employed for the
present purpose, with pUC13 being especially suitable.
The fusion protein of modified human PSTI and APH can be produced
by introducing the expression plasmids prepared in this manner into
an appropriate microbial host, just as described above with
reference to the manufacture of unmodified human PSTI. Thus, since
the modified human PSTI protein is expressed in the form of a
fusion protein, digestion by the proteases produced by the host
microorganism is avoided. The fusion protein obtained by the above
procedure is cleaved by one of the appropriate methods stated
above, thereby yielding the modified human PSTI. Amino acid
analysis of the modified human PSTI so obtained (Ser(44)-PSTI,
Thr(43)-PSTI and Gln(42)-PSTI) showed that the numbers of the
respective amino acid residues in each of these products differed
in the expected manner from those of the original PSTI (Example 2,
Table 2). Investigation of the trypsin-inhibiting activity of each
variety of modified human PSTI revealed that, in the case of
Gln(42)-PSTI and Ser(44)-PSTI, the temporary trypsin inhibition
observed in the case of natural human PSTI was diminished, and in
fact the persistence of inhibitory effect upon trypsin was actually
prolonged as compared with natural human PSTI (Example 2, FIGS. 3
and 4). This demonstrated that the present invention provides
modified human PSTI with superior characteristics.
BRIEF DESCRIPTION OF THE DRAWINGS
This invention may be better understood and its numerous objects
and advantages will become apparent to those skilled in the art by
reference to the accompanying drawings as follows:
FIG. 1 is the amino acid sequence of the modified human PSTI
Gln(42)-PSTI of the present invention and the DNA encoding the
same.
FIG. 2 is the amino acid sequence of the modified human PSTI
Ser(44)-PSTI of the present invention and the DNA encoding the
same.
FIG. 3 is of a graph showing the comparative stability under
trypsin treatment at pH 7.0 of natural human PSTI and the three
varieties of modified human PSTI (viz, Ser(44)-PSTI, Thr(43)-PSTI
and Gln(42)-PSTI) of the present invention.
FIG. 4 is of a graph showing the comparative stability under
trypsin treatment at pH 8.0 of natural human PSTI and the three
varieties of modified human PSTI (viz, Ser(44)-PSTI, Thr(43)-PSTI
and Gln(42)-PSTI) of the present invention.
FIG. 5 is the amino acid sequence of natural human PSTI and the DNA
encoding the same.
FIG. 6-1, 6-2 and 6-3 show the DNA sequence of the APH gene and the
amino acid sequence deduced from this DNA sequence.
FIG. 7 is the DNA sequence of the synthetic human PSTI gene used in
the present invention and the amino acid sequence corresponding to
this DNA sequence.
FIG. 8 is a restriction endonuclease map showing the recognition
sites of various restriction enzymes within and in the vicinity of
the APH gene.
FIG. 9 is an explanatory diagram which schematically indicates the
essential features of the procedure for constructing the expression
plasmid pUC13-PSTI, containing the DNA sequence encoding the fusion
protein of APH and natural human PSTI, which is used in the present
invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
EXAMPLE 1
Construction and Expression of DNA Encoding Natural Human PSTI
The DNA sequence of the natural human PSTI gene determined by
Yamamoto et al. (Biochem. Biophys. Res. Commun., 132, 605, (1985))
was assumed. The DNA sequence of the structural gene encoding this
mature protein of human PSTI was synthesized, the methionine codon
ATG was ligated to the 5' end of this sequence, and the termination
codon TAG was 1ligated to the 3' end. Then, this DNA sequence with
both start and stop codons was further augmented by additional base
sequences in such a manner that the resulting sequence possesses a
recognition site for the restriction enzyme AccI at the 5' end as
well as a recognition site for the restriction enzyme BamHI at the
3' end, the design being such that a double stranded molecule with
single-strands of base length 179 and 181 is formed.
Firstly, in order to prepare the above DNA fragment, the inventors
chemically synthesized 20 short-chain DNA fragments comprising two
groups, i.e., one group which, if ligated in the proper order,
would form a DNA chain including the sequence encoding the amino
acid sequence of human PSTI (U-1 to U-10, FIG. 7), and another
group which, if suitably ligated, would form the complementary
sequence to this DNA chain (L-1 to L-10, FIG. 7). These fragments,
if all the varieties are mixed together, can form double stranded
structures with mutually complementary fragments joined by hydrogen
bonds and having cohesive ends which constitute recognition sites
for restriction endonucleases as described above (FIG. 7).
The above 20 varieties of short-chain DNA fragments (U-1 to U-10
and L-1 to L-10) were prepared, using an automatic nucleic acid
synthesizer (GENETOA-II, manufactured by Nippon Zeon Co.). Each of
the fragments so obtained was purified by gel chromatography using
Sephadex G-50 and reverse phase high performance liquid
chromatography with a silica gel column (Nucleosil 10C18,
10,/.mu.m, 10 .times.250 mm).
Since the 20 oligonucleotides synthesized in this manner possess no
phosphate group at the 5' terminus, they cannot be joined by T4 DNA
ligase as they stand. Therefore using an enzymatic addition
reaction, phosphate groups were attached to the 5' termini of
eighteen of these twenty varieties of synthetic oligonucleotides,
viz, all except U-1 and L-10. This phosphorylation reaction was
effected with T4 polynucleotide kinase (manufactured by Takara
Shuzo Co.). Approximately 300 pmol of each oligonucleotide was
dissolved in 25 .mu.l of the kinase reaction solution (50 mM Tris
hydrochloride buffer, 10 mM magnesium chloride, 10 mM
2-mercaptoethanol, app. 1000 pmol ATP, pH 7.6), then the reaction
was initiated by adding 3 units of T4 polynucleotide kinase to the
solution and continued for 1 hour at 37.degree. C. Then, after heat
treatment of the reaction solution at 65.degree. C for 20 minutes
to inactivate the T4 polynucleotide kinase, the solution was used
directly for the ligation reaction. Then, 50 pmol of each of the
eighteen varieties of phosphorylated synthetic oligonucleotides U-2
to U-10 and L-1 to L-9 as well as the two unphosphorylated
synthetic oligonucleotides U-1 and L-10 were mixed to prepare a
reaction solution for ligation, which was first heat-treated at
80.degree. C for 2 minutes and then slowly cooled down to
20.degree. C. Next, dithiothreitol, ATP and T4 DNA ligase were
added, and the ligation reaction was conducted for 5 days at
4.degree. C. The final composition of this ligation reaction
solution (200 .mu.l) was 66 mM Tris hydrochloride buffer, 66 mM
magnesium chloride, 10 mM dithiothreitol, 1 mM ATP and 700 units T4
DNA ligase (Takara Shuzo Co.). These operations were basically
performed in accordance with the procedure described in Nucleic
Acids Res. 13, 2959 (1985). After the ligation reaction, phenol
extraction and ethanol precipitation were carried out in the usual
manner, after which the desired DNA fragment with approximately 180
base pairs was separated by polyacrylamide gel electrophoresis
using a Tris borate buffer solution. The DNA fractionated on the
gel was stained with ethidium bromide, and a DEAE membrane
(Schlleicher and Schuell Co.) was inserted into the gel in the
vicinity of the target DNA band. Next, the said DNA was recovered
by electrophoretically adsorbing the DNA band onto the DEAE
membrane. After the migration of the DNA band toward the DEAE
membrane had been completed, the DNA was eluted from the said
membrane using a solution containing 1.0 M sodium chloride, 10 mM
Tris hydrochloride buffer (pH 8.0) and 1.0 mM EDTA, and recovered
from the eluent by ethanol precipitation. The procedure used here
is a general one, details of which are described, for example, in
"Molecular Cloning" (Cold Spring Harbor Laboratory, New York,
250-251, 1982).
For the purpose of DNA sequencing, the DNA fragments recovered in
this manner were inserted into an M13 phage vector. To accomplish
this, first, the M13mplO phage vector (Takara Shuzo Co.) was
cleaved with the restriction enzymes AccI and BamHI to form a
linear chain, which was then joined, using T4 DNA ligase, to the
DNA fragment which had been recovered as described above. The
ligation reaction was conducted under virtually the same conditions
as the previously described one for ligation of synthetic
oligonucleotides, except that the reaction temperature and time in
the prevent case were 12.degree. C. and 16 hours, respectively.
After ligation, the DNA so treated was used for the transformation
of a E. coli host in accordance with the method described in
"Molecular Cloning" (Cold Spring Harbor Laboratory, New York,
250-251, 1982).
DNA recipient bacteria obtained from a culture of E. coli K12
strain JMI03 in the logarithmic growth phase by treatment with
calcium chloride at 0.degree. C. were mixed with the DNA ligated by
the above-described reaction, and the mixture was incubated in ice,
after which transformation was effected by heat treatment at
42.degree. C. for 2 minutes. The E. coli cells transfected with the
M13mp10 phage were detected as plaques by the following method.
First, the JMI03 bacteria were added to a mixture of 20 .mu.l of
100 mM isopropyl-.beta.-D-thiogalactoside, 50 .mu.l of 2%
5-bromo-4-chloro-3-.beta.-galactoside, 0.2 ml of a suspension of
JM103 in the logarithmic growth phase and 3 ml of soft agar (0.6%
liquid agar), and this was poured onto 1.5% agar plates. The agar
used here contained TY culture medium (8 g trypton, 5 g yeast
extract and 5 g sodium chloride dissolved in 1 liter of water).
After overnight incubation at 37.degree. C., the transformed
bacteria formed plaques. The bacteria transformed by the M13mp10
phage into which the desired DNA fragments had been inserted
(referred to below as M13-PSTI) formed colorless plaques, whereas
those bacteria transformed by M13mp10 without the desired DNA
insertions formed blue plaques.
Single-stranded phage DNA was prepared from the aforesaid colorless
plaques in accordance with the method of Messing (Methods Enzymol.
101, 20-28 (1983)), using the following procedure. 1 ml of a
culture solution containing E. coli K-12 strain JM103 incubated for
one night was placed in 100 ml of 2xTY medium (viz, 16 g
bactotrypton, 10 g yeast extract and 5 g sodium chloride dissolved
in 1 liter of water) and shake-cultured for 2 hours at 37.degree.
C. This culture solution was divided into 5 ml aliquots, then the
agar where the plaques had formed was aspirated into capillary
pipettes and inoculated into the said culture solution. Next, the
culture solution was incubated for another 5 hours at 37.degree. C.
to induce infection by M13-PSTI and release of phage into the
culture medium. The intact bacterial cells in the culture solution
were used for the preparation of double stranded DNA, while the
supernatant of the culture medium, from which the bacterial cells
had been removed, was utilized for the preparation of
single-stranded phage DNA.
Then, 800.mu.l of 2.5 M sodium chloride solution containing 20%
poly-ethylene glycol was added to 4 ml of the culture medium
supernatant, and the phage was collected by centrifugal separation.
This phage was dissolved in a 500 .mu.l of a solution composed of
10 mM Tris hydrochloride buffer (pH 8.0) and 1 mM
ethylenediaminetetraacetic acid (EDTA), after which the
single-stranded DNA was recovered by phenol extraction and ethanol
precipitation. Replicable double stranded circular DNA was prepared
from the phage-infected bacteria in accordance with the
conventional sodium hydroxide-sodium dodecyl sulfate (SDS) method
(Nucleic Acids Res. 7, 1513-1523 (1979)) by the following
procedure. First, the bacterial cells obtained from 5 ml of culture
liquid were suspended in 100 .mu.l of 25 mM Tris hydrochloride (pH
8.0, containing 50 mM glucose, 10 mM EDTA and 4 mg/ml lysozyme),
and left at room temperature for 5 minutes. To this was added
200.mu.l of 0.2 M sodium hydroxide solution containing 1% SDS, and
after gentle mixing the suspension was left in ice for 5 minutes.
Then, 150.mu.l of 5 M potassium acetate solution (pH 5.2 ) was
added, and after mixing the suspension was again left in ice for at
least 5 minutes. Next, after centrifugation, two volumes of ethanol
were added to one volume of the supernatant fluid and the
precipitate was recovered. This precipitate was then washed with
70% ethanol, and again recovered by centrifugation. In this manner,
replicable double stranded DNA was prepared from the colorless
plaques. This DNA was then cleaved at two sites by AccI and BamHI,
and formation of DNA fragments with approximately 180 base pairs
was verified. Next, the single-stranded phage DNA prepared from the
same plaques was used for base sequencing by the dideoxy method
(Science 214, 1205-1210 (1981)). Base sequencing was performed with
an M13 Sequencing Kit (Takara Shuzo Co.). In this manner, it was
verified that the cloned DNA so obtained did indeed include the
entire structural gene for the desired PSTI. After verification of
the base sequence, the replicable double stranded DNA was used for
the construction of a PSTI expression plasmid, as follows.
The PSTI expression plasmid was constructed by joining the
following three fragments.
1) The AccI-BamHI fragment of approximately 180 bp with verified
base sequence obtained by the above-described ligation reaction of
synthetic oligonucleotides.
2) The approximately 2.8 kbp DNA fragment resulting from cleavage
of pUC13 (Takara Shuzo Co.) by HindIII and BamHI.
3) The approximately 600 bp DNA fragment obtained by digesting pNEO
(containing the APH gene of Tn5; Pharmacia Co.) with HindIII,
followed by digestion with TaqI (corresponding to the DNA sequence
from position -350 to position 246 in FIG. 6; see FIG. 8).
Among these, the DNA fragments 1) and 3) were separated by
polyacrylamide gel electrophoresis, recovered with a DEAE membrane
and used for the subsequent ligation reaction. The above-mentioned
fragment 2), after verification of cleavage at the two specified
sites, was recovered by phenol extraction and ethanol precipitation
and then used for the ligation reaction. The PSTI expression
plasmid (pUC13-PSTI) obtained by the ligation of these three
fragments expresses a fusion protein consisting of PSTI joined at a
site 82nd residues downstream from the amino terminus of the APH
encoded in the transposon Tn5. As in the case previously described,
T4 DNA ligase was employed for the ligation of these three
fragments. The DNA obtained by the ligation reaction was used for
transformation in accordance with the method described in
"Molecular Cloning" (v.s.). Transformation was performed using E.
coli K-12 strain C600 or AG-1 that was used as the DNA recipient.
Since the transformed bacteria acquire ampicillin resistance,
phenotypic selection was performed with reference to the formation
of colonies on agar plates containing ampicillin (with LB culture
medium, viz, 10 g trypton, 5 g yeast extract and 5 g sodium
chloride in 1 liter of water). Using a platinum loop, 12 of the
colonies so formed were transplanted to 5 ml of LB culture medium
containing 40 .mu.g/ml ampicillin and incubated at 37.degree. C.
for 16 hours. Then, the bacteria were collected by centrifugation
and the plasmids were analyzed by the previously described sodium
hydroxide-SDS method. Since the target plasmid (pUC13-PSTI)
contains just one recognition site for each of the restriction
enzymes HindIII, BamHI and PstI, this plasmid can be detected by
the formation of an approximately 3.6 kb DNA band upon digestion
with each of these enzymes.
The clones which were verified as possessing the desired plasmid
were cultured in LB medium (containing 40 .mu.g/ml ampicillin) and
then stored at -70.degree. C. in the presence of 50% glycerol.
Then, 10,.mu.l of this bacterial stock solution was added to 5 ml
of LB medium containing ampicillin and incubated at 37.degree. C.
for 8 hours. Then, 100 .mu.l of this culture liquid was added to 5
ml of M9 culture medium containing ampicillin (M9 medium was
prepared by dissolving 6 g disodium hydrogenphosphate, 3 g
potassium dihydrogenphosphate, 0.5 g sodium chloride and 1 g
ammonium chloride in 1 liter of water, and after sterilization,
adding magnesium sulfate and calcium chloride in quantities such
that their final concentrations are 2 mM and 0.1 mM, respectively;
in addition, the medium contained 40 .mu.g/ml ampicillin, 0.5%
glucose and 0.5% casamino acids), and incubation was continued for
24 hours at 37.degree. C. After the incubation was completed, the
bacteria were collected by centrifugation and used for the
following analytical procedure.
A small quantity of bacteria was taken as a sample for analysis by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). The bacterial protein was dissolved in a liquid
composed of 0.1 M Tris hydrochloride buffer (pH 6.8), 1% SDS, 1%
2-mercaptoethanol and 20% glycerol, extracted and subjected to gel
electrophoretic analysis. The fusion protein containing PSTI
appeared as a major band at the position corresponding to the
expected molecular weight 15,000, thus confirming the expression of
this fusion protein in these transformed E. coli. In addition,
samples of these transformed E. coli were lysed by methods such as
ultrasonication and the protein content of the bacteria was
separated into soluble and insoluble fractions by centrifugation;
SDS-PAGE analysis of these fractions revealed that the said fusion
protein existed mainly in the insoluble protein fraction.
Then, 6 g of these bacteria were suspended in 20 ml of 0.1 M Tris
hydrochloride solution (pH 7.0, containing 5 mM EDTA), and
centrifuged at 12,000 .times.g for 10 minutes. After repeating the
same operation, the bacteria were again suspended in 15 ml of 0.1 M
Tris hydrochloride solution (pH 7.0, containing 5 mM EDTA, 50 mM
benzamidine and 1 mM phenylmethanesulfonyl fluoride (PMSF)), and
then crushed three times with a French press at a pressure of 400
kg/sq.cm. Then, 1.05 g of the pellet obtained by 20 minutes of
centrifugation at 23,000 .times.g was dissolved in 10 ml of 0.1 M
sodium phosphate (pH 7.0, containing 20 mM dithiothreitol (DTT) and
a protein denaturing agent), and this was subjected to gel
filtration on a Sephacryl S-200 column (2.6.times.79 cm), followed
by elution with 0.1 M Tris hydrochloride (pH 7.2, containing 1 mM
DTT and 7 M urea). The fraction with a molecular weight of
approximately 17,000 daltons was collected, dialyzed against
distilled water, and lyophilized. To the lyophilate, 2 ml of 70%
formic acid solution containing 160 mg of cyanogen bromide was
added, and the mixture was allowed to react for 6 hours at room
temperature. Then, 18 ml of distilled water was added thereto and
the sample was again lyophilized. The lyophilate so obtained was
dissolved in 2 ml of 0.5 M Tris hydrochloride (pH 8.1, containing 2
mM EDTA and 6 M guanidine hydrochloride) and 100 .mu.l of
2-mercaptoethanol was added. After reacting for 4 hours at
37.degree. C. under a nitrogen stream, the mixture was dialyzed
against distilled water. The sample was then centrifuged at 10,000
x g for 1 minute, to 6 ml of the supernatant fluid so obtained was
added 172 mg sodium chloride and 320 .mu.l of 1 M Tris
hydrochloride (pH 8.0), and the sample was adsorbed onto an
affinity column (2.times.3 cm) charged with bovine
trypsin-CH-Sepharose 4B. This column was then washed successively
with 0.05 M Tris hydrochloride (pH 8.0) containing 0.5 M sodium
chloride and with distilled water, after which the PSTI was eluted
with 10 mM hydrochloric acid, followed by lyophilization, resulting
a purified substance of 1.55 mg.
Then, 12 .mu.g of the human PSTI so obtained was placed in test
tube (10 x 90 mm), of 4 M methanesulfonic acid (containing 0.2% of
3-(2-aminoethyl)indole) was added, and the sample was hydrolyzed
under reduced pressure at 110.degree. C. for 24 hours. This
hydrolysate was then subjected to amino acid analysis using a
Hitachi Model 835 amino acid analyzer; the results so obtained are
given in Table 1, indicating that the amino acid composition of the
PSTI obtained by the process described above was completely
identical with that of natural PSTI (theoretical values). Also,
investigation of the amino acid sequence of the three residues at
the N-terminus by the method of Edman (modification of the method
of Iwanaga et al., Eur. J. Biochem. 8, 189-199, 1969) revealed that
this was Asp-Ser-Leu, i.e., identical with that of natural human
PSTI. Moreover, the human PSTI obtained by the present method
inhibited bovine trypsin in the stoichiometric molar ratio 1:1, and
furthermore, the results of immunological reaction with antibody
raised against natural human PSTI (rabbit antiserum polyclonal
antibody) were the same as those observed in the case of natural
human PSTI (i.e., the behavior of the dilution curve was identical
with t hat of natural human PSTI).
TABLE 1 ______________________________________ Experimental
Theoretical Amino acid value value
______________________________________ Asp 7.8 8 Thr 3.8 4 Ser 2.8
3 Glu 6.2 6 Pro 2.9 3 Gly 5.2 5 Ala 1.4 1 1/2Cys 5.6 6 Val 2.0 2
Met 0.0 0 Ile 2.8 3 Leu 4.0 4 Tyr 2.9 3 Phe 1.2 1 Lys 3.8 4 His 0.0
0 Trp 0.0 0 Arg 3.0 3 ______________________________________
EXAMPLE 2
Construction of DNA Sequence Encoding Modified Human PSTI;
Expression of Said Modified Human PSTI by Escherichia coli and
Purification thereof
1. Preparation of Ser(44)-PSTI
Preparation of this modified human PSTI (Ser(44)-PSTI) was effected
by preparing, as a template, the single-stranded recombinant
M13-APH/PSTI which includes the gene encoding a fusion protein of
APH and PSTI, and then introducing thereinto a sitespecific
mutation using the synthetic DNA oligomer to be described below as
primer. These operations were performed in accordance with the
procedure indicated in the manual for the Amersham
oligonucleotide-directed in vitro mutagenesis system. a)
Preparation of single-stranded DNA containing gene coding for
fusion protein of APH and PSTI
Bacteria were collected by centrifugation from 5 ml of a liquid LB
medium containing a culture of E. coli which had been transformed
with the PSTI expression plasmid pUC13-PSTI, obtained in the manner
described in Example 1, and the said plasmids were recovered by the
sodium hydroxide-SDS method. These pUC13-PSTI plasmids were then
dissolved in 20 .mu.l of 10 mM Tris hydrochloride (pH 8.0,
containing 1 mM EDTA), and were cleaved by a reaction with the
restriction enzymes HindIII and BamHI at 37.degree. C. for 1.5
hours. The DNA fragments so obtained were separated by agarose
electrophoresis and recovered with a DEAE membrane. Using T4 DNA
ligase, these DNA fragments were then spliced to phage M13mp10
which had been cleaved with HindIII and BamHI, thereby constructing
a recombinant (M13-APH/PSTI) carrying DNA which encodes the
APH-PSTI fusion protein. Using this recombinant, E. coli K-12
strain JMI03 cells were transformed under the same conditions as
those used in Example 1 above, except that the duration of heat
treatment at 42.degree. C. after the treatment at 0.degree. C. was
done for 1.5 minutes. The JM103 bacteria into which M13-APH/PSTI
had been introduced were incubated overnight on agar plates at
37.degree. C. in the same manner as in Example 1, and using 2 ml of
this overnight culture liquid, single-stranded DNA was prepared
from the bacteria which had formed colorless plaques.
b) Synthesis of primer
The DNA fragment represented by the following base sequence (1) was
synthesized with a GENET A-II automatic nucleic acid synthesizer
(Nippon Zeon Co.) for use as a primer in site-specific mutagenesis.
This DNA fragment includes the sequence encoding the amino acid
sequence from the 41st to the 46th residues of the modified human
PSTI in which the arginine in position 44 of natural human PSTI has
been replaced by serine. ##STR2##
The DNA fragment so obtained was purified by gel chromatography
using Sephadex G-50 and by reverse phase high performance liquid
chromatography with silica gel (Nucleosil C18; 10 .mu.m,
10.times.250 mm).
c) Site-specific mutagenesis in vivo
First, 200 pmol of the DNA fragment (1) purified in above item b)
was dissolved in 100 mM Tris hydrochloride buffer solution (pH 7.6,
containing 10 mM magnesium chloride, 10 mM DTT and 0.5 mM ATP), and
was phosphorylated by a reaction with 10 units of T4 polynucleotide
kinase (PL biochemical) at 37.degree. C for 1 hour. Then, the T4
polynucleotide kinase was inactivated by heat treatment at
65.degree. C for 10 minutes. Next, in 17.mu.l of 5-fold diluted
Buffer Solution 1 (Amersham), 5 pmol of this phosphorylated DNA
fragment was annealed with 1.5 pmol of the single-stranded
recombinant (M13-APH/PSTI) obtained in above item a). This reaction
was effected by heating for 10 minutes at 70.degree. C. followed by
incubation for 30 minutes at 37.degree. C. Then, to this 17.mu.l
annealed mixture were added 5 .mu.l of 100 mM magnesium chloride,
19 .mu.l of Nucleotide Mix 1 (Amersham), 6.mu.l of water, 1.6 .mu.l
of DNA polymerase I Klenow fragment (3.8 units/.mu.l) and 2.4 .mu.l
of T4 DNA ligase (2.5 units/.mu.l), and double stranded DNA was
synthesized by allowing this mixture to react overnight (19-21
hours) at 16.degree. C. Next, the residual single-stranded DNA in
this mixture, which had not been converted into double strands, was
removed by a nitrocellulose filter. Then, 0.1 volumes of 3 M
ammonium acetate and 2.5 volumes of ethanol were added to the
solution containing the double-stranded DNA, the precipitated DNA
was then dissolved in 25 .mu.l of Buffer Solution 2 (Amersham), to
10.mu.l of this solution were added 65.mu.l of Buffer Solution 3
(Amersham) and 0.7.mu.l of restriction enzyme NciI (8 units/.mu.l),
and the mixture was allowed to react for 90 minutes at 37.degree.
C. Then, to 65.7 .mu.l of this reaction mixture were added 12 .mu.l
of 500 mM sodium chloride, of Buffer Solution 4 (Amersham) and of
exonuclease III (25 units/.mu.l), and the mixture was allowed to
react for 28 minutes at 37.degree. C. This was then heat-treated at
70.infin. C. for 15 minutes to terminate the enzymatic reaction.
Next, 5.mu.l of 100 mM magnesium chloride, 13.mu.l of Nucleotide
Mix 2, 0.86.mu.l of DNA polymerase I (3.5 units/.mu.l) and 0.8
.mu.l of T4 DNA ligase (2.5 units/.mu.l) were added, and a reaction
was conducted at 16.degree. C. for 4 hours. In this manner, double
stranded DNA containing the gene for Ser(44)-PSTI was prepared, and
used for the following transformation.
A DNA recipient obtained by calcium chloride treatment at 0.degree.
C. of a culture solution of E. coli K-12 strain JMI03 in the
logarithmic growth phase was mixed with the above-mentioned double
stranded DNA carrying the Ser(44)-PSTI gene. This mixture was
incubated at 0.degree. C. for 20 minutes and then heat-treated at
42.degree. C. for 1.5 minutes to effect the transformation of the
bacteria.
The JM103 bacteria into which the above-mentioned DNA had been
introduced were cultured on agar plates and single-stranded DNA was
prepared from the bacteria which produced colorless colonies in the
same manner as described in the preceding Example 1. The
preparation of replicable double-stranded circular DNA from
phage-infected bacteria was also performed by the
sodium-hydroxide-SDS method in the same manner as was done in
Example 1. That is, the bacteria obtained from 4 ml of culture were
suspended in 100.mu.l of 25 mM Tris hydrochloride (pH 8.0,
containing 50 mM glucose, 10 mM EDTA and 4 mg/ml lysozyme), and the
sample was left at room temperature for 5 minutes. Then, 200 .mu.l
of 0.2 M sodium hydroxide containing 1% SDS was added, and after
gentle mixing the sample was left in ice for 5 minutes. Next,
150.mu.l of 5 M potassium acetate solution (pH 5.2) was added, and
after mixing the sample was left in ice for at least 10 minutes.
Then, after centrifuging, replicable double-stranded DNA was
recovered from the supernatant by phenol extraction followed by
ethanol precipitation. Then, the single-stranded phage DNA prepared
from the same plaque was subjected to DNA base sequencing by the
dideoxy method, in the same manner as indicated in Example 1 above,
and the sequencing results verified that the clone obtained by the
present procedure did indeed contain the complete base sequence of
the structural gene for the desired modified PSTI (Ser(44)-PSTI).
The replicable double-stranded DNA, the base sequence of which had
been verified in this manner, was then used for the construction of
the following expression plasmid. d) Construction of expression
plasmid
Approximately 3.5 .mu.g of the replicable double-stranded DNA
obtained in above item c) was cleaved with the restriction
endonucleases EcoRI and HindIII, and then the EcoRI/HindIII
fragment was separated by polyacrylamide gel electrophoresis and
recovered with a DEAE membrane. Using T4 ligase, this DNA fragment
(containing the gene (approximately 700 bp) encoding the desired
APH/Ser(44)-PSTI fusion protein) was ligated with the plasmid pUC13
(Takara Shuzo Co.) which had been cleaved with EcoRI and HindIII,
thereby constructing the expression plasmid pUC13(Ser(44)-PSTI).
Using this plasmid, E. coli recipients were transformed by the
method indicated in "Molecular Cloning" (v.s.).
e) Expression of Ser(44)-PSTI
Transformation was performed using E. coli K-12 strain C600 or AG-1
as a DNA recipient. Since the transformed bacteria acquire
ampicillin resistance, phenotypic selection was performed with
reference to formation of colonies on agar plates containing
ampicillin (with LB medium, viz, 10 g trypton, 5 g yeast extract
and 5 g sodium chloride in 1 liter of water). Using a sterilized
bamboo skewer, eight of the colonies so formed were transplanted
into 5 ml of LB medium containing 100.mu.g/ml ampicillin which was
then incubated 18 hours at 37.degree. C. Then, the bacteria were
collected by centrifugation and the plasmids were recovered in the
same manner as described in item c) above.
The clones which had been verified as (possessing the desired
plasmid pUC13(Ser(44)-PSTI) were preserved at -70.degree. C. in the
presence of 50% glycerol. Then, 0.1 ml of this bacterial stock was
added to 100 ml of LB medium containing 100 /.mu.g/ml ampicillin,
and this culture was incubated overnight at 37.degree. C.. Next,
37.5 ml of this culture was added to 1.5 liters of LB medium
containing 100 .mu.g/ml ampicillin, and this was further incubated
for one night at 37.degree. C.. After this incubation was
completed, the bacteria were collected by centrifugation and stored
at -20.degree. C..
f) Purification of Ser(44)-PSTI
2.2 g of the bacteria obtained in above item d) were suspended in
10 ml of 0.1 M Tris hydrochloride (pH 7.0, containing 5 mM EDTA),
and the suspension was centrifuged at 12,000.times.g for 10
minutes. After repetition of the same operation, the bacteria were
suspended in 10 ml of 0.1 M Tris hydrochloride (pH 7.0, containing
50 mM benzamidine and 1 mM PMSF), and this suspension was crushed 3
times under a pressure of 400 kg/sq.cm with a French press. Then,
0.44 g of the pellet obtained by centrifuging this sample for 30
minutes at 23,000.times.g was dissolved in 10 ml of 0.1 M sodium
phosphate (pH 7.0, containing 8 M guanidine hydrochloride and 20 mM
DTT), and this was subjected to gel filtration with a Sephacryl
S-200 column (2.6.times.79 cm) and eluted with 0.1 M Tris
hydrochloride (pH 7.2, containing 1 mM DTT and 7 M urea). The
fraction of molecular weight approximately 17,000 daltons (35 ml),
corresponding to the desired APH/PSTI fusion protein, was collected
and 20 ml of this fraction was dialyzed against distilled water and
then lyophilized. This lyophilate was dissolved in 0.3 ml of 70%
formic acid, then 200 .mu.l of cyanogen bromide (200 mg/ml) was
added and the mixture was allowed to react at room temperature for
6 hours. Next, 10 times by volume (i.e., 18 ml) of distilled water
was added and this mixture was lyophilized. Then, the lyophilate so
obtained was dissolved in 2 ml of 0.05 M Tris hydrochloride (pH
8.0, containing 0.5 M sodium chloride) and centrifuged at
10,000.times.g for 1 minute, and the supernatant was subjected to
adsorption in an affinity column (1.times.3 cm) charged with bovine
trypsin-CH-Sepharose 4B. This column was then washed successively
with 0.05 M Tris hydrochloride (pH 8.0) and distilled water, after
which the modified PSTI was eluted with 12 mM hydrochloric acid and
lyophilized to obtain 415 .mu.g of the purified substance.
2. Preparation of Gln(42)-PSTI and Thr(43)-PSTI
Gln(42)-PSTI and Thr(43)-PSTI were prepared in a manner similar to
that employed for the preparation of Ser(44)-PSTI, using as
primers, however, the synthetic DNA oligomers indicated in the
following formulae (2) and (3), respectively. ##STR3##
3. Respective properties of three varieties of modified human
PSTI
a) Amino acid composition
With respect to each of the three varieties of modified human PSTI
(Ser(44)-PSTI, Gln(42)-PSTI, and Thr(43)-PSTI) described above,
approximately 10 .mu.g of the substance was placed in a test tube
(10.times.90 mm), to which was then added 50.mu.l of 4 M
methanesulfonic acid (containing 0.2% of 3-(2-aminoethyl)indole),
and the mixture was hydrolyzed under reduced pressure for 24 hours
at 110.degree. C. This sample was then subjected to amino acid
analysis, using a Hitachi Model 835 amino acid analyzer. The amino
acid compositions of each variety of modified PSTI as well as the
theoretical composition of natural human PSTI are shown in Table 2.
As indicated by this table, the numbers of the respective amino
acid residues in each of these varieties of modified human PSTI
differed from those of the original human PSTI in the theoretically
anticipated manner, thereby confirming that the desired varieties
of modified human PSTI had indeed been obtained by the processes
described above.
TABLE 2 ______________________________________ Natural PSTI Amino
Gln(42)- Thr(43)- Ser(44)- (theoretical acid PSTI PSTI PSTI values)
______________________________________ Asp 7.7(8) 7.5(8) 7.8(8) 8
Thr 3.8(4) 4.5(5) 3.7(4) 4 Ser 2.8(3) 2.7(3) 3.5(4) 3 Glu 7.1(7)
6.2(6) 6.4(6) 6 Pro 2.8(3) 2.7(3) 2.9(3) 3 Gly 5.1(5) 5.0(5) 4.9(5)
5 Ala 1.3(1) 1.4(1) 1.3(1) 1 1/2Cys 5.2(6) 5.1(6) 5.0(6) 6 Val
2.1(2) 2.1(2) 2.0(2) 2 Met 0.0(0) 0.0(0) 0.0(0) 0 Ile 2.9(3) 2.7(3)
2.8(3) 3 Leu 4.2(4) 4.0(4) 4.2(4) 4 Tyr 2.9(3) 2.7(3) 2.9(3) 3 Phe
1.1(1) 1.3(1) 1.2(1) 1 Lys 4.0(4) 3.2(3) 3.8(4) 4 His 0.0(0) 0.0(0)
0.0(0) 0 Trp 0.0(0) 0.0(0) 0.0(0) 0 Arg 2.2(2) 3.0(3) 2.1(2) 3
Total 56 56 56 56 ______________________________________
Trypsin inhibitory activity of modified PSTI
Investigation of t he inhibitory activity of each variety of
modified PSTI revealed that each of the said varieties of PSTI
inhibited human trypsin in the stoichiometric molar ratio of 1:1.
Next, the transience of inhibitory effects was investigated with
respect to each modified PSTI. This term transience as used here
refers to the fact that PSTI initially inhibits human trypsin, but
with subsequent passage of time trypsin activity is recovered,
indicating that the PSTI has been inactivated. This phenomenon is
known to occur in the case of natural PSTI.
First, 1 nmol of human trypsin was incubated at 37.degree. C. in
200.mu.l of 0.1 M Tris hydrochloride (pH 7.0 or 8.0, containing 20
mM calcium chloride and 0.004% Triton X-100) together with 2 nmol
of natural human PSTI or one of the three varieties of modified
PSTI obtained by the processes described above. At prescribed times
a 20 .mu.l aliquot of the mixture was removed and placed in a test
tube containing 150.mu.l of 0.5 M Tris hydrochloride (pH 8.0), 200
.mu.l of 5 mM benzoyl-L-arginine p-nitroanilide and 500 .mu.l of
distilled water, and incubated at 37.degree. C. for 5 minutes,
after which the reaction was terminated by adding 500 .mu.l of 30%
acetic acid, then the absorbance at 410 nm was measured and the
trypsin-inhibitory activity was calculated. The results of these
experiments for pH 7.0 and pH 8.0 are indicated in FIGS. 3 and 4,
respectively.
As is clearly shown by FIGS. 3 and 4, for either pH 7.0 and pH 8.0,
the temporary inhibitory action was markedly diminished for both
Gln(42)-PSTI, with Gln replacing Arg at the 42nd position, and
Ser(44)-PSTI, with Ser replacing Arg at the 44th position, as
compared with natural human PSTI; thus, the persistence of activity
as a trypsin inhibitor was actually increased by these
substitutions. In particular, at pH 7.0, Gln(42)-PSTI had
maintained trypsin-inhibiting activity even 24 hours after the
initiation of the reaction. On the other hand, Thr(43)-PSTI, with
Thr replacing Lys at the 43rd position, displayed almost the same
inhibitory transience as natural human PSTI at pH 7.0, while at pH
8.0 this modified PSTI displayed even less persistence of
trysin-inhibiting effect than the natural form.
Thus, the present invention provides DNA sequences encoding
modified varieties of human PSTI possessing excellent stability in
terms of decreased susceptibility to decomposition by proteolytic
enzymes such as trypsin, as compared with natural human PSTI, as
well as the modified varieties of human PSTI obtained by expression
of the said DNA sequences. Since these modified varieties of human
PSTI are produced by recombinant DNA techniques, mass production of
these substances at low prices can be realized. Moreover, since the
amino acid sequences of these substances differ from that of
natural human PSTI only at one position, the clinical application
of these substances entails virtually no danger of allergic
reactions, as compared with the bovine product BPTI and chemically
synthesized agents which have been clinically used as trypsin
inhibitors up until now. Furthermore, since the said varieties of
modified human PSTI are less susceptible to decomposition by
proteolytic enzymes such as trypsin and display more stable and
sustained trypsin-inhibiting action as compared with natural human
PSTI, these new varieties offer the prospect of higher clinical
utility in the treatment of pancreatitis.
It is understood that various other modifications will be apparent
to and can be readily made by those skilled in the art without
departing from the scope and spirit of this invention. Accordingly,
it is not intended that the scope of the claims appended hereto be
limited to the description as set forth herein, but rather that the
claims be construed as encompassing all the features of patentable
novelty that reside in the present invention, including all
features that would be treated as equivalents thereof by those
skilled in the art to which this invention pertains.
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